The present invention relates to the field of molecular biology and genetics. Specifically, the invention relates to the use of recombinant viruses and recombinant viral vectors in expressing foreign peptides and/or regulating gene expression in a plant host.
Virus expression vectors allow rapid expression of host and non-host genes in plant systems. The construction of cDNA libraries within virus expression allows gene function to be assessed by screening plant hosts for phenotypic changes and gain of function effects measured by various input and output focused screening. The ability of virus vectors to effectively reprogram host machinery to favor expression of virally encoded genes has been used to overexpress genes of pharmaceutical relevance in plants. This capacity has moved from being a mere laboratory curiosity, to being exploited at greenhouse and field scale to produce products used in human clinical trials. These expression systems have primarily exploited dicot hosts, especially in the families of Solanaceae and Crucifereace. However, one report has been published for a potyvirus expression vector in monocot plants. This vector will allow the insertion of precise ORFs within the virus polyprotein for the accumulation of only cytosolically localized proteins (Choi, et al., 2000). This type of vector is limited in that only exact insertions are possible in this vector and not allowing insertion of crude cDNAs or libraries. It is further limited by not allowing the expression of secreted proteins in plants.
Continued research on virus expression vectors soon revealed new utilities. It has been extensively demonstrated that virus vectors are efficient tools to induce post-transcriptional gene silencing systemically in host plants. However, these effects have only been demonstrated in dicot hosts, primarily in Nicotiana benthamiana and Arabidopsis thaliana. The power of shuttling in plant gene sequences systemically into plants and screening infected plants for loss of function effects is a unique tool to determine function of endogenes. To confirm the effects of loss of function or gain of function events discovered in dicot plants, the ability to either express genes or silence them in monocot system would provide a powerful tool to confirm gene function in a second system. However, at present, no such tool exists. It is the purpose of this disclosure to describe the invention and reduction to practice of a monocot gene expression and gene silencing system.
Gopinath, et al. (2000) discloses a cowpea mosaic virus expressing a movement protein-green fluorescent protein (xe2x80x9cGFPxe2x80x9d)-large coat protein fusion protein, with the foot and mouth disease virus 2A catalytic peptide at each side of the GFP insert. However, expression of the fusion protein is only demonstrated in a cowpea plant, which is a dicot plant.
WO99/36516 disclose the use of viral vectors to silence endogenous genes in a host, through post-transcription gene silencing of the endogenous host gene homolog, by introducing nucleic acids into the host by way of constructs based on tobacco mosaic virus or tomato mosaic virus.
Vectors for the genetic manipulation of plants have been derived from several naturally occurring plant viruses, including barley stripe mosaic virus (xe2x80x9cBSMVxe2x80x9d). BSMV is a tripartite RNA virus (Palomar, et al., 1977) (FIG. 1A) that infects many agriculturally important monocot species such as oat, wheat and barley (McKinney and Greeley, 1965). We developed several monocot viral vectors based on the BSMV genome that direct the expression of nucleotide sequences in transfected protoplasts and plants.
The present invention is directed to recombinant viruses comprising a viral genome or one or more recombinant viral vectors, each further comprising a polynucleotide encoding a fusion protein or a foreign protein of interest or a targeting nucleotide sequence capable of silencing a gene of interest. The present invention is also directed to a host comprising the recombinant virus, recombinant viral vector, or polynucleotide. The present is also directed to methods of expressing the fusion protein or a foreign protein, or silencing a gene of interest in a host.
(A) One aspect of the present invention is directed to one or more polynucleotide(s), wherein at least one of the one or more polynucleotide encodes a promoter operatively linked to a transcriptional unit, wherein the transcriptional unit encodes a fusion protein, wherein the fusion protein comprises (1) a viral protein, (2) a protein of interest, and (3) autoproteolytic peptide, wherein the autoproteolytic peptide is fused between the viral protein and the protein of interest. In one embodiment, the protein of interest is able to be directed to a specific location within the host cell.
(B) Another aspect of the present invention is also directed to one or more polynucleotide(s), wherein at least one of the one or more polynucleotide(s) encodes a promoter operatively linked to a transcriptional unit, wherein the transcriptional unit encodes a protein of interest, wherein the one or more polynucleotide(s) constitute a viral genome, wherein the gene, or fragment thereof, encoding a coat protein of the viral genome is deleted or mutated, wherein the coat protein is native to the viral genome. The deletion or mutation of the coat protein gene, or fragment thereof, causes an increased, elevated, enhanced, or high expression of the transcription unit when compared to a identical one or more polynucleotide(s) that does not have the deletion or mutation of the coat protein gene, or fragment thereof.
(C) Another aspect of the present invention is also directed to one or more polynucleotide(s), wherein at least one of the one or more polynucleotide(s) encodes a promoter operatively linked to a transcriptional unit, wherein the transcriptional unit encodes a protein of interest, wherein said polynucleotide is a duplicated genomic nucleic acid component. In one embodiment of the invention, there is only one duplicated genomic nucleic acid component.
(D) Another aspect of the present invention is also directed to one or more polynucleotide(s), wherein at least one of the one or more polynucleotide(s) encodes a promoter operatively linked to a transcriptional unit, wherein the transcriptional unit encodes a viral protein, a stop codon, and a targeting nucleotide sequence, wherein the a viral protein is 5xe2x80x2 of the stop codon, and the stop codon is 5xe2x80x2 of the targeting nucleotide sequence, wherein the viral protein, the stop codon, and the targeting nucleotide sequence are transcribed as a transcriptional unit, wherein the targeting nucleotide sequence is substantially homologous to a gene of interest, or fragment thereof, to be silenced. The target nucleotide sequence can be in the sense or antisense orientation of the gene of interest to be silenced. Preferably the target nucleotide sequence is in the sense orientation. Preferably silencing takes place in the cytoplasm.